The determination of blood glucose concentration is extremely important in clinical diagnosis and in the management of diabetes. Approximately 150 million people worldwide suffer from the chronic disease diabetes mellitus, a figure that may double by 2025 according to the WHO. Although diabetes is readily diagnosed and treated, successful long-term management requires low-cost diagnostic tools that rapidly and accurately report blood glucose concentrations. PQQ-dependent glucose dehydrogenases (EC 1.1.5.2) catalyze a reaction in which glucose is oxidized to gluconolactone. Consequently, this type of enzyme is used in measuring blood sugar. One of these tools is a diagnostic strip based on the soluble glucose dehydrogenase (s-GlucDOR, EC 1.1.5.2), a pyrroloquinoline quinone-containing enzyme originally derived from Acinetobacter calcoaceticus. 
Quinoproteins use quinone as cofactor to oxidize alcohols, amines and aldoses to their corresponding lactones, aldehydes and aldolic acids (Duine, J. A., Energy generation and the glucose dehydrogenase pathway in Acinetobacter, in “The Biology of Acinetobacter” New York, Plenum Press (1991), pp. 295-312; Duine, J. A., Eur. J. Biochem. 200 (1991) 271-284; Davidson, V. L., in “Principles and applications of quinoproteins”, the whole book, New York, Marcel Dekker (1993); Anthony, C., Biochem. J. 320 (1996) 697-711; Anthony, C. and Ghosh, M., Current Science 72 (1997) 716-727; Anthony, C., Biochem. Soc. Trans. 26 (1998) 413-417; Anthony, C. and Ghosh, M., Prog. Biophys. Mol. Biol. 69 (1998) 1-22. Among quinoproteins, those containing the noncovalently bound cofactor 2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quinoline-4,5-dione (PQQ) constitute the largest sub-group (Duine 1991, supra). All bacterial quinone glucose dehydrogenases known so far belong to this sub-group with PQQ as cofactor (Anthony and Ghosh 1997 supra; Goodwin, P. M. and Anthony, C., Adv. Microbiol. Physiol. 40 (1998) 1-80; Anthony, C., Adv. in Phot. and Resp. 15 (2004) 203-225).
Two types of PQQ-dependent glucose dehydrogenase (EC 1.1.5.2) have been characterized in bacteria: One is membrane-bound (m-GDH); the other is soluble (s-GDH). Both types do not share any significant sequence homology (Cleton-Jansen, A. M., et al., Mol. Gen. Genet. 217 (1989) 430-436; Cleton-Jansen, A. M., et al., Antonie Van Leeuwenhoek 56 (1989) 73-79; Oubrie, A., et al., Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 11787-11791. They are also different regarding both their kinetic as well as their immunological properties (Matsushita, K., et al., Bioscience Biotechnol. & Biochem. 59 (1995) 1548-1555). The m-GDHs are widespread in Gram-negative bacteria, s-GDHs, however, have been found only in the periplasmatic space of Acinetobacter strains, like A. calcoaceticus (Duine, J. A., 1991a; Cleton-Jansen, A. M. et al., J. Bacteriol. 170 (1988) 2121-2125; Matsushita and Adachi, 1993) and A. baumannii (JP 11243949).
Through searching sequence databases, two sequences homologous to the full-length A. calcoaceticus s-GDH have been identified in E. coli K-12 and Synechocystis sp. Additionally, two incomplete sequences homologous to A. calcoaceticus s-GDH were also found in the genome of P. aeruginosa and Bordetella pertussis (Oubrie et al. 1999 a, b, c) and Enterobacter intermedium (Kim, C. H. et al., Current Microbiol. 47 (2003) 457-461), respectively. The deduced amino acid sequences of these four uncharacterized proteins are closely related to A. calcoaceticus s-GDH with many residues in the putative active site absolutely conserved. These homologous proteins are likely to have a similar structure and to catalyze similar PQQ-dependent reactions (Oubrie et al., 1999 a, b, c; Oubrie A., Biochim. Biophys. Acta 1647 (2003) 143-151; Reddy, S., and Bruice, T. C., J. Am. Chem. Soc. 126 (2004) 2431-2438; Yamada, M. et al., Biochim. Biophys. Acta 1647 (2003) 185-192).
Bacterial s-GDHs and m-GDHs have been found to possess quite different sequences and different substrate specificity. For example, A. calcoaceticus contains two different PQQ-dependent glucose dehydrogenases, one designated m-GDH which is active in vivo, and the other designated s-GDH for which only in vitro activity can be shown. Cleton-Jansen et al., 1988; 1989 a, b cloned the genes coding for the two GDH enzymes and determined the DNA sequences of both of these GDH genes. There is no obvious homology between m-GDH and s-GDH corroborating the fact that m-GDH and s-GDH represent two completely different molecules (Laurinavicius, V., et al., Biologija (2003) 31-34).
The gene of s-GDH from A. calcoaceticus has been cloned in E. coli. After being produced in the cell, the s-GDH is translocated through the cytoplasmic membrane into the periplasmic space (Duine, J. A., Energy generation and the glucose dehydrogenase pathway in Acinetobacter, in “The Biology of Acinetobacter”, New York, Plenum Press (1991), pp. 295-312; Matsushita, K. and Adachi, O., Bacterial quinoproteins glucose dehydrogenase and alcohol dehydrogenase, in “Principles and applications of Quinoproteins”, New York, Marcel Dekker (1993) pp. 47-63). Like the native s-GDH from A. calcoaceticus, recombinant s-GDH expressed in E. coli is a homodimer, with one PQQ molecule and three calcium ions per monomer (Dokter, P. et al., Biochem. J. 239 (1986) 163-167; Dokter, P. et al., FEMS Microbiol. Lett. 43 (1987) 195-200; Dokter, P. et al., Biochem. J. 254 (1988) 131-138; Olsthoorn, A. J. and Duine, J. A., Arch. Biochem. Biophys. 336 (1996) 42-48; Oubrie, A., et al., J. Mol. Biol. 289 (1999) 319-333; Oubrie, A., et al., Proc. Natl. Acad. Sci. U.S.A 96 (1999) 11787-11791; Oubrie, A., et al., Embo J. 18 (1999) 5187-5194). s-GDH oxidizes a wide range of mono- and disaccharides to the corresponding ketones which further hydrolyze to the aldonic acids, and it is also able to donate electrons to PMS (phenazine metosulfate), DCPIP (2,6-dichlorophenolindophenol), WB (Wurster's blue) and short-chain ubiquinones such as ubiquinone Q1 and ubiquinone Q2 (Matsushita, K., et al., Biochem. 28 (1989) 6276-6280; Matsushita, K., et al., Antonie Van Leeuwenhoek 56 (1989) 63-72), several artificial electron acceptors such as N-methylphenazonium methyl sulfate (Olsthoom, A. J. and Duine, J. A., Arch. Biochem. Biophys. 336 (1996) 42-48; Olsthoorn, A. J. and Duine, J. A., Biochem. 37 (1998) 13854-13861) and electro conducting polymers (Ye, L., et al., Anal. Chem. 65 (1993) 238-241). In view of s-GDH's high specific activity towards glucose (Olsthoom, A. J. and Duine, J. A., (1996) supra) and its broad artificial electron acceptor specificity, the enzyme is well suited for analytical applications, particularly for being used in (bio-)sensor or test strips for glucose determination in diagnostic applications (Kaufmann, N. et al., Development and evaluation of a new system for determining glucose from fresh capillary blood and heparinized blood in “Glucotrend” (1997) 1-16, Boehringer Mannheim GmbH; Woosuck, S. et al., Sensors and Actuators B 100 (2004) 395-402).
Glucose oxidation can be catalyzed by at least three quite distinct groups of enzymes, i.e., by NAD/P-dependent glucose dehydrogenases, by flavoprotein glucose oxidases or by quinoprotein GDHs (Duine, J. A., Biosens. Bioelectronics 10 (1995) 17-23). A rather slow autooxidation of reduced s-GDH has been observed, demonstrating that oxygen is a very poor electron acceptor for s-GDH (Olsthoom and Duine, 1996). s-GDH can efficiently donate electrons from the reduced quinone to mediators such as PMS, DCPIP, WB and short-chain ubiquinones such as Q1 and Q2, but it can not efficiently donate electrons directly to oxygen.
Traditional test strips and sensors for monitoring glucose level in blood, serum and urine e.g. from diabetic patients use glucose oxidase. The performance of the enzyme is dependent of the oxygen concentration. Glucose measurements at different altitudes with different oxygen concentrations in the air may lead to false results. The major advantage of PQQ-dependent glucose dehydrogenases is their independence from oxygen. This important feature is e.g., discussed in U.S. Pat. No. 6,103,509, in which some features of membrane-bound GDH have been investigated.
An important contribution to the field has been the use of s-GDH together with appropriate mediators. Assay methods and test strip devices based on s-GDH are disclosed in detail in U.S. Pat. No. 5,484,708. This patent also contains detailed information on the set-up of assays and the production of s-GDH-based test strips for measurement of glucose. The methods described there as well as in the cited documents are herewith included by reference.
Other patents or applications relating to the field and comprising specific information on various modes of applications for enzymes with glucose dehydrogenase activity are U.S. Pat. No. 5,997,817; U.S. Pat. No. 6,057,120; EP 0 620 283; and JP 11-243949-A.
A commercial system which utilizes s-GDH and an indicator that produces a color change when the reaction occurs (Kaufmann, et al., 1997, supra) is the Glucotrend® system distributed by Roche Diagnostics GmbH.
Despite the above discussed advantages for use of a PQQ dependent s-GDH, in the determination of glucose also a disadvantage has to be considered. The enzyme has rather a broad substrate spectrum as compared to m-GDH. That is, s-GDH oxidizes not only glucose but also several other sugars including maltose, galactose, lactose, mannose, xylose and ribose (Dokter et al. 1986 a; Oubrie A., Biochim. Biophys. Acta 1647 (2003) 143-151). The reactivity towards sugars other than glucose may in certain cases impair the accuracy of determining blood glucose levels. In particular patients on peritoneal dialysis, treated with icodextrin (a glucose polymer) may contain in their body fluids, e.g., in blood, high levels of other sugars, especially of maltose (Wens, R., et al., Perit. Dial. Int. 18 (1998) 603-609).
Therefore clinical samples as e.g. obtained from diabetic patients, especially from patients with renal complications and especially from patients under dialysis may contain significant levels of other sugars, especially maltose. Glucose determinations in samples obtained from such critical patients may be impaired by maltose (Frampton, J. E. and Plosker, G. L., Drugs 63 (2003) 2079-2105).
There are few reports in the literature on attempts to produce modified PQQ-dependent s-GDHs with altered substrate specificity. Igarashi, S., et al., Biochem. Biophys. Res. Commun. 264 (11999) 820-824 report that introducing a point mutation at position Glu277 leads to mutants with altered substrate specificity profile.
Sode, EP 1 176 202, reports that certain amino acid substitutions within s-GDH lead to mutant s-GDH with an improved affinity for glucose. In EP 1 167 519 the same author reports on mutant s-GDH with improved stability. Furthermore the same author reports in JP2004173538 on other s-GDH mutants with improved affinity for glucose.
Kratzsch, P. et al., WO 02/34919 report that the specificity of s-GDH for glucose as compared to other sugar substrates, especially as compared to maltose, can be improved by amino acid substitutions in certain positions of s-GDH. Central and crucial is a substitution at amino acid position 348. A mutant s-GDH comprising for example a glycine in position 348 instead of a threonine as present in the wild-type s-GDH has a tremendously improved selectivity for the substrate glucose as, e.g. as compared to the substrate maltose. They also disclose that a double mutant having substitutions at positions 348 and 428 have an even more improved specificity for glucose.
In WO 2006/008132 it is shown that an amino acid insertion between amino acids 428 and 429 of s-GDH, especially in combination with an appropriate amino acid substitution at position 348 has quite favorable effects on substrate specificity. Mutants comprising this insertion are for example more specific for the substrate glucose as compared to the substrate maltose.
However, whereas quite some improvements on glucose specificity have been reported, it appears that such improvements frequently and unfortunately go hand in hand with disadvantages like e.g. a reduced stability, a reduced activity and/or a reduced affinity for glucose of such mutated s-GDH. For example, it has become evident that the improved specificity of an s-GDH mutant comprising an amino acid substitution in position 348 goes to the expense of stability, affinity and activity of said mutant as compared to the wild-type enzyme.
A great demand and clinical need therefore exists for further improved mutant forms of s-GDH having a high specificity for glucose and which feature at the same time a reasonable thermo stability, as well as improvements in specific activity or affinity for glucose, or that feature improvements in both specific activity and affinity for glucose.
It was the task of the present invention to provide new mutants or variants of s-GDH with significantly improve thermo stability, specific activity and affinity for glucose as compared to a mutant with improved specificity comprising a substitution at position 348.
It has been found that it is possible to significantly improve the thermo stability, the specific activity and the affinity for glucose of an s-GDH mutant having a substitution at position 348 by selecting mutations from the positions as given in the appending claims.
Due to the improved properties of the new forms of s-GDH, significant technical progress for glucose determinations in various fields of applications is possible. The improved s-GDH mutants according to this invention can for example be used with great advantage for the specific detection or measurement of glucose in biological samples.